Effects of dietary limonene and thymol on the growth and nutritional physiology of Nile tilapia (Oreochromis niloticus)

Phytogenic compounds such as limonene and thymol have been shown to have growth-promoting properties in farmed animals but studies in fish are scarce. Two Experiments (Experiments I and II) were carried out to investigate the individual effects of dietary limonene and thymol on the growth and nutritional physiology of juvenile Nile tilapia (Oreochromis niloticus). In Experiment I, the fish were fed on a commercial diet coated with limonene at 0 (control), 200, 400, and 600 mg kg-1 (ppm), while in Experiment II thymol was supplemented in the diet at 0 (control), 250 and 500 ppm. Our results showed a significant increase in fish weight and weight gain with diets supplemented with 400 and 600 ppm limonene compared to the control. Moreover, the expression of insulin growth factor I (igf-I), mucin-like protein (muc), oligo-peptide transporter I (pept1), lipoprotein lipase (lpl), alkaline phosphatase (alp) and catalase (cat) was up-regulated by dietary limonene. Our results confirm that dietary limonene can enhance the growth of Nile tilapia juveniles through the activation of key genes involved in somatotropic axis-mediated growth, nutrient digestion and antioxidant enzyme defence. Dietary thymol did not seem to influence growth or regulate the same pathways activated by limonene in Nile tilapia juveniles at inclusion levels up to 500 ppm. Overall, the present results suggest that potential growth-promoting effects are dependent upon the phytogenetic itself and its inclusion level

Delta-8 desaturation activity varies among fatty acyl desaturases of teleost fish: high activity in delta-6 desaturases of marine species

The benefits of dietary fish and fish oil are derived from n-3 long-chain polyunsaturated fatty acids (LC-PUFA) that have beneficial effects in a range of human diseases and pathologies such as cardiovascular and other inflammatory disorders, neural development and neurological pathologies. The precursor of n-3 LC-PUFA, 18:3n-3 does not have the same beneficial effects prompting interest in the pathways of endogenous synthesis of LC-PUFA in vertebrates. The LC-PUFA biosynthesis pathway classically involves Δ6 and Δ5 fatty acyl desaturases (Fad), but it was recently shown that Δ6 Fad in mammals also displayed Δ8 activity demonstrating a possible alternative “Δ8-pathway” for the synthesis of LC-PUFA. Our primary hypothesis was that Δ8 desaturase activity would be a common feature of vertebrate Δ6 Fads, and so the aim of the present study was to determine the ability of teleostei Fads for Δ8 desaturation activity. To this end, cDNAs for Fads from a range of freshwater, diadromous and marine teleost fish species were assayed for Δ8 activity in the heterologous yeast expression system. In summary, the present study has demonstrated that Δ8 desaturation activity was also a characteristic of fish orthologs, although the activity varied notably between freshwater/diadromous and marine fish species, with the latter possessing Fads2-like proteins with Δ8 activity far higher than mammalian FADS2. The data showed that, generally, the fish Fad are technically υ-3 desaturases, with new double bonds introduced 3C beyond a pre-existing double bond. However, the ability of zebrafish and rabbitfish Fads, previously characterised as Δ6/Δ5 bifunctional desaturases, to introduce non-methylene interrupted double bonds in 20:3n-3 and 20:2n-6 suggested that a novel combination of regioselectivity modes operates within these enzymes

Future availability of raw materials for salmon feeds and supply chain implications: the case of Scottish farmed salmon

The current range of Scottish salmon feeds is adapted to a differentiated supply of salmon products, including differing omega-3 content, differing content of marine raw materials, etc. The progressive replacement of marine feed ingredients by plant proteins and oils is reducing the content of omega-3 long-chain polyunsaturated fatty acids (LC-PUFA). However the benefits are a more secure and less volatile raw material supply, together with environmental feed contaminants at low or undetectable levels in the resulting salmon product. There is widespread adoption of standards and certification schemes by Scottish salmon farmers and feed suppliers in order to demonstrate environmental sustainability. This has focused in particular on use of certified ingredients from sustainable supply sources (‘responsible sourcing’). Future volume estimates of Scottish salmon production, hence feed requirements, are insufficient to threaten raw material supply compared with global markets, although it is argued this is likely to involve greater use of locally grown plant proteins and an increased proportion of fishmeal manufactured from by-product trimmings (derived from processing fish for human consumption). However, UK retail chains will remain reluctant to allow salmon suppliers to utilise land animal by-products due to negative consumer perceptions, with resulting implications for formulation cost and flexibility. Given its world-wide scarcity, the main strategic concern relates to future availability of sufficient omega-3 LC-PUFA, in particular eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), in order to maintain the healthy image of Scottish salmon. To maintain its longer-term reputation and product benefits, the Scottish industry may need to consider adopting a more flexible attitude to using new alternatives to fish oil (e.g. EPA and DHA derived from transgenic oil seed crops, when commercially available). It is concluded that Scottish salmon farming is a successful example of sustainable feed development and the industry can be confident that the changing raw material base will support continuing production of high quality, healthy farmed salmon, but the long-term security of supply of omega-3 LC-PUFA remains an issue

Long-chain polyunsaturated fatty acid biosynthesis in chordates: insights into the evolution of Fads and Elovl gene repertoire

Long-chain polyunsaturated fatty acids (LC-PUFA) are major components of complex lipid molecules and are also involved in numerous critical biological processes. Studies conducted mainly in vertebrates have demonstrated that LC-PUFA can be biosynthesized through the concerted action of two sets of enzymes, namely fatty acyl desaturases (Fads) and elongation of very long-chain fatty acid (Elovl) proteins. While LC-PUFA research is a thriving field, mainly focused on human health, an integrated view regarding the evolution of LC-PUFA biosynthetic genetic machinery in chordates is yet to be produced. Particularly important is to understand whether lineage specific life history trajectories, as well as major biological transitions, or particular genomic processes such as genome duplications have impacted the evolution of LC-PUFA biosynthetic pathways. Here we review the gene repertoire of Fads and Elovl in chordate genomes and the diversity of substrate specificities acquired during evolution. We take advantage of the magnitude of genomic and functional data to show that combination duplication processes and functional plasticity have generated a wide diversity of physiological capacities in extant lineages. A clear evolutionary framework is provided, which will be instrumental for the full clarification of functional capacities between the various vertebrate groups

Biosynthesis of long-chain polyunsaturated fatty acids in the African catfish Clarias gariepinus: Molecular cloning and functional characterisation of fatty acyl desaturase (fads2) and elongase (elovl2) cDNAs7

Fish differ in their capacity for endogenous synthesis of long-chain (C20-24) polyunsaturated fatty acids (LC-PUFA) from dietary C18 precursors (α-linolenic and linoleic acids). Understanding this capacity is of benefit to fish feed formulation. This, together with the importance of fish as the primary source of omega-3 LC-PUFA in the human diet has necessitated the rigorous study of the biochemical and molecular mechanisms involved in the LC-PUFA biosynthesis pathway in fish species. Studies have shown the potential of a species for LC-PUFA biosynthesis is associated with the complement and function of fatty acyl desaturase (fads) and elongase of very long chain fatty acid (elovl) gene it possesses. The present study therefore aimed to investigate these genes in the African catfish (Clarias gariepinus), the most commercially important farmed fish species in Sub-Saharan Africa. A fads2 and an elovl2 cDNA were cloned containing open reading frames (ORF) of 1338 base pair (bp) and 864 bp specifying proteins of 445 and 287 amino acids, respectively. Functional characterisation by heterologous expression in yeast showed that the Fads2 was bifunctional with ∆5∆6 activities catalysing the desaturation of both 18:3n-3 and 20:4n-3 and their corresponding n-6 fatty acids, 18:2n-6 and 20:3n-6. The Elovl2 showed activity towards C18, C20 and C22 PUFA with highest activity towards C20 and C22 PUFA. Tissue expression analysis showed a typical freshwater species expression pattern; higher expression in the liver compared to brain and all other tissues with the exception of elovl5 which showed highest expression in the intestine. Consistent with feeding studies of typical freshwater fish species that show their essential fatty acid requirement can be satisfied by dietary C18 PUFA, the present study confirms that the LC-PUFA biosynthesis pathway is active in the African catfish C. gariepinus

The effects of combined phytogenics on growth and nutritional physiology of Nile tilapia Oreochromis niloticus

This study investigated whether dietary supplementation of phytogenic compounds limonene and thymol had synergistic or additive effects on growth and selected nutritional physiology pathways in Nile tilapia. A 63-day feeding experiment was conducted using fish of 1.5 ± 0.0 g (± standard error) fed on a commercial diet coated with either 0 ppm limonene and thymol (control), 400 ppm limonene (L), 500 ppm thymol, (T) or a combination of 400 ppm limonene and 500 ppm thymol (LT). Final fish weight (FW) was significantly improved to similar extents by diet LT (16.7 ± 0.3 g) and L (16.6 ± 0.4 g). Dietary thymol alone and the control did not enhance FW (15.0 ± 0.4 g and 13.7 ± 0.4 g respectively). Dietary thymol had shown a strong tendency to improve somatic growth (P =.052). The analysed candidate genes involved in the pathways of nutrient digestion, absorption and transport (muc), lipid metabolism (lpl), antioxidant enzymes (cat) and somatotropic axis growth (igf[sbnd]I) were also up-regulated to similar extents in Nile tilapia by diet L and LT (P < .05), above the regulation observed with the diet supplemented exclusively with thymol. This suggests lack of synergistic or additive effects on growth and nutritional physiology pathways when limonene and thymol are supplied in the diet

Growth performance, nutrient utilisation and body composition of Macrobrachium rosenbergii fed graded levels of phytic acid

Information relating to the effects of phytic acid (PA), an anti-nutritional factor (ANF) commonly found in plant protein ingredients, on the growth performance, feed conversion ratio (FCR), nutrient utilisation and whole proximate composition in the Malaysian giant freshwater prawn, Macrobrachium rosenbergii, is lacking. Replicate groups of M. rosenbergii (mean initial carapace length of 6.03 &plusmn; 0.30 mm; mean initial weight of 0.29 &plusmn; 0.02 g; n = 20 per replicate group) were fed graded levels of PA for 140 days. The basal diet, to which different levels of PA were added to obtain 0.26 (control), 6.48, 11.28, 16.53, 21.45 and 26.16 g PA kg-1, contained fishmeal, soy protein concentrate and wheat meal. Terminal sampling indicated that the growth performance, FCR and survival did not differ significantly between the groups receiving the different inclusions of PA within their diets. The apparent protein, lipid and energy utilisation responded negatively, decreasing significantly (p&lt;0.05) with an increasing inclusion of PA, particularly within the groups of prawns fed diets with the highest inclusions of PA (i.e. 21.45&ndash;26.16 g PA kg-1). The whole body composition of protein (p&lt;0.04), lipid (p&lt;0.01) and gross energy (p&lt;0.05) also decreased significantly with an increasing supplementation of PA, while the ash content significantly increased (p&lt;0.01), most notably in the groups of prawns receiving the highest levels of dietary PA (26.16 g PA kg-1). In conclusion, inclusions of up to 11.28 g PA kg-1 appear safe but levels above this have negative impacts. The results show that PA had significant effects on the nutrient utilisation and body compositions (p&lt;0.05) but not on growth performance and FCR (p&gt;0.05) on the M. rosenbergii reared under the experimental conditions that were employed here

The compositional and metabolic responses of gilthead seabream (Sparus aurata) to a gradient of dietary fish oil and associated n-3 long-chain PUFA content

The authors express their gratitude to the technical team at the BioMar Feed Trial Unit, Hirtshals, in particular, Svend Jørgen Steenfeldt for expert care of the experimental subjects, for training and supervision provided by laboratory staff at Nutrition Analytical Services and Molecular Biology at the Institute of Aquaculture, University of Stirling, UK. S. J. S. H’s. PhD was co-funded by BioMar and the Marine Alliance for Science and Technology Scotland. BioMar provided the experimental feeds, trial facilities and fish, and covered travel expenses. V. K. and J. T. designed and executed the nutritional trial and all authors contributed to planning the analyses. V. K., J. T. and S. J. S. H. carried out the sampling. O. M., D. R. T and S. A. M. M. supervised the lead author. M. B. B. provided training in molecular biology to S. J. S. H. who carried out all analytical procedures. S. J. S. H. analysed all of the data and prepared the manuscript. Subsequently the manuscript was shared between all authors who made amendments, contributions and recommendations. The authors declare that there are no conflicts of interestPeer reviewedPublisher PD

Essential fatty acid metabolism and requirements of the cleaner fish, ballan wrasse Labrus bergylta: Defining pathways of long-chain polyunsaturated fatty acid biosynthesis

Ballan wrasse (Labrus bergylta) is an effective counter-measure against sea lice used by Atlantic salmon farmers, proving to be more effective and economical than drugs or chemical treatments alone. There are currently efforts underway to establish a robust culture system for this species, however, essential fatty acid dietary requirements are not known for ballan wrasse. In the present study, we isolated and functionally characterised ballan wrasse fatty acid desaturase (Fads) and elongation of very long-chain fatty acids (Elovl) protein to elucidate their long-chain polyunsaturated fatty acid (LC-PUFA) biosynthetic capability. Sequence and phylogenetic analysis demonstrated that the cloned genes were fads2 and elovl5 orthologues of other teleost species. Functional characterisations of fads2 and elovl5 were performed using the yeast (Saccharomyces cerevisiae) heterologous expression system. The Fads2 showed &Delta;6 desaturase activity towards 18:3n&ndash;3, 18:2n&ndash;6 and 24:5n&ndash;3, and &Delta;8 desaturase activity towards 20:3n&ndash;6 and 20:2n&ndash;6. The Elovl5 showed elongase activities towards various C18 and C20 fatty acids. Therefore, 20:4n&ndash;3 and 20:3n&ndash;6 can be synthesised from 18:3n&ndash;3 and 18:2n&ndash;6, respectively in ballan wrasse via two possible pathways, the &Delta;6 (&Delta;6 desaturation &ndash; elongation) and &Delta;8 (elongation &ndash; &Delta;8 desaturation) pathways. However, due to the absence of &Delta;5 desaturase activity and no other Fads2 in their genome, 20:5n&ndash;3 (eicosapentaenoic acid, EPA) and 20:4n&ndash;6 (arachidonic acid, ARA) cannot be synthesised from C18 PUFA precursors and they could consequently be regarded as dietary essential fatty acids for ballan wrasse. Since no &Delta;4 desaturase activity was detected in ballan wrasse Fads2, 22:6n&ndash;3 (docosahexaenoic acid, DHA) can only be synthesised from EPA via the Sprecher pathway comprising two sequential elongation steps to produce 24:5n&ndash;3 followed by &Delta;6 desaturation and chain shortening. Although ballan wrasse Elovl5 had no elongase activity towards C22, other elongases such as Elovl4 exist in the ballan wrasse genome that may be able to produce 24:5n&ndash;3. Therefore, as ballan wrasse Fads2 can desaturate 24:5n&ndash;3 to produce 24:6n-&shy;3, it can be assumed that ballan wrasse can synthesise DHA from EPA

Biosynthesis of essential fatty acids in Octopus vulgaris (Cuvier, 1797): Molecular cloning, functional characterisation and tissue distribution of a fatty acyl elongase

Polyunsaturated fatty acids (PUFA) have been identified as key nutrients for the common octopus (Octopus vulgaris), particularly for its early life-cycle stages (paralarvae). Our overarching aim is to establish the essential fatty acid (FA) requirements for octopus paralarvae through determination of the enzymes of endogenous PUFA biosynthetic pathways. We here report on the molecular cloning and functional characterisation of a cDNA encoding a putative elongase of very long-chain fatty acids (Elovl), a critical enzyme that mediate the elongation of FA including PUFA. Our results suggested that the octopus Elovl is phylogenetically related to Elovl5 and Elovl2, two elongases with demonstrated roles in PUFA biosynthesis in vertebrates. Further evidence supporting a role of the octopus Elovl in PUFA biosynthesis was provided through functional characterisation of its activity in yeast. It was confirmed that expression of the octopus Elovl conferred on yeast the ability to elongate some C18 and C20 PUFA, while C22 PUFA substrates remained unmodified. The substrate specificities exhibited by the octopus elongase were consistent with those of vertebrate Elovl5. Interestingly, the octopus Elovl elongated n-6 PUFA substrates more efficiently than their analogous n-3 substrates, suggesting that n-6 PUFA may have particular biological significance in O. vulgaris. Finally, we investigated the potential role of the newly cloned Elovl in the biosynthesis of non-methylene-interrupted FA, compounds typically found in marine invertebrates and confirmed to be also present in the common octopus